• Immunofluorescence (if-test)

The IF-test is commonly used to identify the unknown antigen in the specimen.

NOTE: IF-test is also known as Coons’ reaction.

The special diagnostic antibodies labeled with some fluorochromes are employed in this test. Fluorescent dyes (e.g., fluorescein, rhodamine) can be covalently attached to antibody molecules and made visible by ultraviolet light in the fluorescence microscope. The antibodies labeled by FITC (or RITC) retain the immunological specificity and interact with strictly definite antigens. Such labeled antibody can be used to identify antigens both on the surface of bacteria and in the cells in histologic section or other specimens. Labeled antibody-antigen complexes are readily recognized by intense yellow-green fluorescence (for FITC) during examination with the fluorescent microscopy. The antibodies labeled by RITC in specific antigen-antibody complexes induce the orange-red fluorescence under the fluorescent microscope. There are several variants of IF-test:

1. A direct immunofluorescence reaction occurs when known labeled antibody interacts directly with unknown antigen. If the antigen is present in the specimen, the antibody will react, and the antibody-coated microbes will glow when observed with the fluorescent microscope.

2. An indirect immunofluorescence reaction occurs when a 2-stages process is used. This variant of IF-test can be performed for detection of antibody or antigen.

3. An anti-complement immunofluorescence reaction is used in cases where the antigen-antibody complex can fix complement. The microbe is fixed to a slide and mixed with unlabeled specific antiserum and guinea pig complement. After washing anti-complement fluorescent-labeled antibodies are added. The labeled antibodies will bind only if the first reaction produced antigen-antibody-complement complexes, which happens only if the antibody in the antiserum is specific for the unknown microbe on the slide.

4. To detect the unknown antibodies in the patient’s serum can be used «sandwich» technique. To do this, the specific known antigen is attached to a slide. Then patient’s serum with “unknown antibodies” is added, and the preparation is washed. If the unknown serum antibody matches the antigen, it will remain fixed to it on the slide and can be detected by adding a fluorescent-labeled antibody and examining the slide by ultraviolet (fluorescent) microscopy.

An indirect immunofluorescence reaction is most commonly used in microbiology. This method is based on the using of two types of antisera. To identify the unknown antigen the unlabeled antibodies against the antigen to be assayed are applied on the slide with the specimen (containing the “unknown” suspected antigen). The preparation is thoroughly washed. At the second stage, the formed antigen-antibody complex is treated with the labeled antiserum containing antibodies against immunoglobulins in the first (unlabeled) antiserum. In this case, antiglobulin antiserum, labeled with fluorochromes coats the unlabeled antibody-unknown antigen complex. And as a result, the antigen becomes visible under the fluorescent microscope. The indirect test is often more sensitive than a direct test, because more labeled antibody adheres to antigenic site. Furthermore, the labeled antiglobulin becomes a «universal reagent» (independent of the nature of the antigen used) reactive with all Ig G of that species (e.g., human Ig G antibodies).